US20120100551A1 - Nucleic acid amplification reaction device, substrate used for nucleic acid amplification reaction device, and nucleic acid amplification reaction method - Google Patents

Nucleic acid amplification reaction device, substrate used for nucleic acid amplification reaction device, and nucleic acid amplification reaction method Download PDF

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US20120100551A1
US20120100551A1 US13/267,980 US201113267980A US2012100551A1 US 20120100551 A1 US20120100551 A1 US 20120100551A1 US 201113267980 A US201113267980 A US 201113267980A US 2012100551 A1 US2012100551 A1 US 2012100551A1
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light
nucleic acid
acid amplification
reaction
amplification reaction
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Kensuke Kojima
Junji Kajihara
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Sony Corp
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Sony Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Definitions

  • the present disclosure relates to nucleic acid amplification reaction devices, substrates used for nucleic acid amplification reaction devices, and nucleic acid amplification reaction methods, and particularly to a nucleic acid amplification reaction device including a reflective component to reflect side light in a reaction area serving as the reaction field of a nucleic acid amplification reaction.
  • the nucleic acid amplification reaction such as the PCR requires a step of checking whether or not the target nucleic acid is specifically amplified.
  • the check is performed by subjecting the reaction liquid used for a nucleic acid amplification reaction such as the PCR to gel electrophoresis by use of a gel of e.g. polyimide and thereafter staining DNA fragments obtained by the PCR amplification.
  • the following related-art methods are also used as a method for checking nucleic acid amplification in a nucleic acid amplification reaction: a method of checking amplification by measuring the turbidity of the reaction liquid used for a nucleic acid amplification reaction; a method of using a microarray including a probe specifically coupled to the nucleic acid as the amplification subject; and real-time PCR in which amplification is checked in real time by using a fluorescently labeled probe coupled to a double-stranded DNA or a fluorescently labeled probe specifically coupled to the target PCR product.
  • the nucleic acid amplification reaction such as the PCR is used also for analysis of e.g. a single nucleotide polymorphism (SNP) and the above-described methods for checking nucleic acid amplification are used.
  • SNP single nucleotide polymorphism
  • SNP analysis can be performed rapidly and easily, e.g. personalized medicine to diagnose the optimum treatment method, medication method, and so forth at the bedside of a patient or the like is enabled and a competent POC (Point Of Care) technique is established.
  • POC Point Of Care
  • a method for checking nucleic acid amplification after nucleic acid amplification reaction more rapidly and easily is desired.
  • a nucleic acid quantification method real-time PCR
  • a method for detecting a variant such as a single nucleotide polymorphism (SNP) (melting curve analysis) are known.
  • SNP single nucleotide polymorphism
  • a probe whose fluorescence intensity changes between the hybridized state (including also a state of being cut after being hybridized) and the free state is used as the fluorescently labeled hybridization probe used in these methods, and detection is performed by measuring this change.
  • a representative thereof is a probe utilizing fluorescence resonance energy transfer (FRET) and TaqMan (trademark) probe and molecular beacon are known as examples of such a probe.
  • nucleic acid probe that utilizes a phenomenon that the light emission of a fluorescent dye decreases when a nucleic acid probe labeled with the fluorescent dye is hybridized with the target nucleic acid and uses one kind of fluorescent dye for the purpose of quantifying a nucleic acid more easily (refer to Japanese Patent Laid-open No. 2005-261354 and Japanese Patent Laid-open No. 2002-119291). Furthermore, regarding a probe labeled with Alexa flour (registered trademark) 350, 488, 568; Pacific Blue (registered trademark), and Cy3, it is also known that the light emission of a fluorescent dye increases in some cases when the labeled probe is hybridized (refer to Marras SAE, Kramer FR, and Tyagi S. (2002), Nucleic Acids Research, 30, e122).
  • the following methods are known as a detecting method in which an electrophoresis gel, a support body such as a film, and a labeled substance are not used.
  • the above-described detecting methods are frequently used, many disadvantages are also found in detection of nucleic acid amplification.
  • many probes have small difference between the excitation wavelength and the fluorescence wavelength, i.e. small Stokes shift, although fluorescence sensitization when the probe is intercalated into a double-stranded nucleic acid in nucleic acid detection is large. Therefore, there are also many disadvantages in terms of crosstalk and the gain.
  • the detection based on precipitation of the magnesium pyrophosphate has an aspect that the signal recognition performance and the appeal power are somewhat poor, although it is extremely easy and practical.
  • nucleic acid amplification reaction device that is easy to use and allows achievement of high detection sensitivity, a substrate used for a nucleic acid amplification reaction device, and a nucleic acid amplification reaction method.
  • a nucleic acid amplification reaction device including a reaction area configured to serve as a reaction field of a nucleic acid amplification reaction, an irradiating unit configured to irradiate light to the reaction area, and a light detecting unit configured to detect the amount of reflected light.
  • a reflective component that reflects side light generated in the reaction area due to light irradiation from the irradiating unit and guides the light to the light detecting unit is disposed.
  • a substrate including a reflective component configured to reflect side light from a reaction area serving as a reaction field of a nucleic acid amplification reaction.
  • a nucleic acid amplification reaction method including guiding side light that is generated due to light irradiation and is from a reaction area serving as a reaction field of a nucleic acid amplification reaction into a light output surface direction and/or a light incident surface direction by a reflective component disposed around the reaction area, and detecting the amount of guided light by a light detector.
  • the embodiments of the present disclosure provide a nucleic acid amplification reaction device, a substrate, and a nucleic acid amplification reaction method that are easy to use and allow achievement of higher detection sensitivity.
  • FIG. 1 is a conceptual diagram of a nucleic acid amplification reaction device according to an embodiment of the present disclosure (first embodiment);
  • FIGS. 2A and 2B show an example of a section of an area around a reaction area along the light incident surface direction-light output surface direction in a substrate according to an embodiment of the present disclosure, and the optical path therein;
  • FIG. 3 is a perspective view of the area around the reaction area in the substrate according to the embodiment of the present disclosure
  • FIGS. 4A to 4C show examples of the substrate around the reaction area according to the embodiment of the present disclosure
  • FIGS. 5A to 5G simply show manufacturing procedures of the substrate according to the embodiment of the present disclosure
  • FIGS. 6A to 6F show examples of a method for fabricating a resin mold used for manufacturing of the substrate according to the embodiment of the present disclosure.
  • FIG. 7 is a conceptual diagram of a nucleic acid amplification reaction device according to an embodiment of the present disclosure (second embodiment).
  • FIG. 1 is a conceptual diagram of a nucleic acid amplification reaction device 1 according to an embodiment of the present disclosure (first embodiment).
  • FIGS. 2A and 2B show a sectional view of an area around a reaction area along the light incident surface direction-light output surface direction in a substrate according to an embodiment of the present disclosure, and an example of the optical path therein.
  • FIG. 3 is a perspective view of the area around the reaction area in the substrate according to the embodiment of the present disclosure.
  • FIGS. 4A to 4C show examples of the substrate around the reaction area according to the embodiment of the present disclosure.
  • the nucleic acid amplification reaction device includes a reaction area 2 , an irradiating unit 3 , and a light detecting unit 5 for controlling a nucleic acid amplification reaction to amplify and quantify a nucleic acid, and is provided with a temperature control unit arbitrarily.
  • a temperature control unit 4 and the reaction area 2 (substrate 6 ) that is detachable are disposed between the irradiating unit 3 and the light detecting unit 5 .
  • a pinhole 7 , an excitation filter 8 , and a collecting lens 9 may be disposed between the reaction area 2 and the irradiating unit 3 arbitrarily in order to adjust the amount of light, the light component, and so forth.
  • a fluorescent filter 10 and a collecting lens 11 may be disposed between the reaction area 2 and the light detecting unit 5 arbitrarily in order to adjust the amount of light, the light component, and so forth.
  • the nucleic acid amplification reaction device 1 of the embodiment of the present disclosure be provided with a controller (not shown) to control respective kinds of operation relating to the device of the embodiment of the present disclosure (e.g. light control, temperature control, nucleic acid amplification reaction, light detection control, calculation of the amount of detected light, and monitoring).
  • a controller not shown to control respective kinds of operation relating to the device of the embodiment of the present disclosure (e.g. light control, temperature control, nucleic acid amplification reaction, light detection control, calculation of the amount of detected light, and monitoring).
  • the reaction area 2 is an area serving as the reaction field of an amplification reaction of a nucleic acid and is disposed at such a position that it can be irradiated with light from the irradiating unit 3 (see FIGS. 1 , 2 A, 2 B, and 7 ).
  • a nucleic acid amplification product is generated in this reaction area 2 in association with the progression of an amplification reaction, and light is generated toward the lateral side of the reaction area 2 when this nucleic acid amplification product is irradiated with light from the irradiating unit 3 .
  • a reflective component 20 is so disposed that this side light is guided to the light detecting unit 5 (see FIGS. 1 , 2 A, 2 B, and 7 ).
  • the shape of the reaction area 2 is not particularly limited as long as an area serving as the reaction field of an amplification reaction of a nucleic acid is provided inside.
  • Examples of the shape include cylindrical shape, conical frustum shape, pyramidal frustum shape (e.g. rectangular frustum shape), and cubic shape.
  • the reflective component 20 is not particularly limited as long as it is so disposed as to reflect the side light from the reaction area 2 and guide the light to the light detecting unit 5 finally (see FIGS. 1 and 7 ). It is preferable that, as shown in FIGS. 2A and 2B , the reflective component 20 (reflective surface 201 ) be so disposed around the reaction area 2 as to guide the side light from the reaction area into the light output surface direction and/or the light incident surface direction for example.
  • the reflection direction of the side light may be adjusted by utilizing plural reflective components 20 (reflective surfaces 201 ) (not shown).
  • the side light may be reflected substantially horizontally by one reflective surface and subsequently the side light may be reflected into the light output surface direction and/or the light incident surface direction by another reflective surface.
  • the reflective surface 201 of the reflective component 20 when the reflective component 20 is cut along the light incident surface direction-light output surface direction may be any surface as long as it is an inclined surface capable of reflecting the side light.
  • the inclined surface include flat surface, curve, and flat surface partially having a curve (see e.g. FIGS. 2A , 2 B and 4 A to 4 C).
  • ⁇ obtuse angle
  • an angle ⁇ in the range of 90 degrees ⁇ 150 degrees is preferable (see FIGS. 2A and 2B ).
  • the three-dimensional shape of the reflective component 20 may be any shape as long as the reflective component 20 can efficiently guide the side light into the light output surface direction and/or the light incident surface direction (see FIGS. 3 and 4A to 4 C).
  • Examples of the three-dimensional shape include trumpet shape, conical frustum shape, and pyramidal frustum shape.
  • the whole surface (all) of the three-dimensional shape may be used for reflection of the side light, or part (one block) of the whole surface of the reflective component 20 may be used.
  • the three-dimensional shape may be divided into plural blocks and each block may be used.
  • the output direction of the side light may be changed on each divided block basis.
  • the blocks may be disposed in such a manner that one block can output light in the light output surface direction and the other blocks can output light in the light incident surface direction.
  • Such a block may be disposed singularly or plurally around the reaction area arbitrarily.
  • the material for light reflection by the reflective component 20 may be any material as long as the material provides high reflectance of the side light.
  • the material include one or more kinds of metal film materials selected from silver, gold, aluminum, rhodium, etc. Among them, silver and a material composed mainly of silver are preferable. By ion sputtering with use of this material, a single-layer or multiple-layer metal film to reflect the side light can be formed as the reflective component 20 (reflective surface 201 ). It is enough that the thickness of the metal film is, but not particularly limited to, about 30 to 200 nm and the thickness per one layer of the metal film is about 30 to 70 nm.
  • the S/N ratio is low and sufficient determination is difficult.
  • providing the above-described reflective component makes it easy to extract the side light from the reaction area and thereby enhances the projected surface area.
  • the turbidity detecting system almost no side scattering is present at the measurement initial stage, at which the amount of nucleic acid amplification product (scattering object) is small, and therefore the sufficiently-high S/N ratio can be ensured from the measurement initial stage if this side scattered light is used as the basis.
  • the determination is also easy from the measurement initial stage and therefore the detection sensitivity can be enhanced although the configuration is simple.
  • the light detecting unit 5 (light receiver) can be disposed on the side of the light incident surface or the light output surface arbitrarily. Therefore, in flexibility of disposing of the optical system and flexibility of the mounting form of the optical system, employing the above-described reflective component is advantageous in terms of the spatial design.
  • a sidewall part 21 is provided between the reaction area 2 and the reflective component 20 , and this sidewall part 21 is in contact with the reaction area 2 at a sidewall 22 (see FIGS. 2A to 4C ).
  • the sidewall part 21 (sidewall 22 ) may be divided into plural blocks (see FIG. 3 ).
  • the sidewall part 21 or each block thereof is formed of a material that transmits or blocks the side light from the reaction area 2 or a material that transmits the necessary light component depending on the purpose.
  • the sidewall part 21 e.g. part between the sidewall 22 and the reflective component 20
  • part thereof may be a space or this space may be filled with a plastic material (e.g. material having no specific wavelength selectivity).
  • the material having no wavelength selectivity may be any material as long as at least scattered light and fluorescence are transmitted through the material. Examples of the material include exemplified materials having optical transparency to be described later.
  • part (block) of the sidewall part 21 a material utilizing a substance that blocks (absorbs) light or a plastic material or the like containing such a substance may be used so that unnecessary side light having an influence in light detection may be blocked.
  • the sidewall part 21 (sidewall 22 ) containing the above-described phosphor material may be used as the phosphor component 23 in order to turn the side light to a light component having the desired specific wavelength.
  • the detachable reaction area 2 (substrate 6 ) having the phosphor component 23 makes it possible to easily extract a light component having the desired specific wavelength (e.g. fluorescence) depending on the method for detecting the nucleic acid amplification product. Moreover, reducing reflected light and an unnecessary light component (e.g. stray light of scattered light) is also permitted. The detection sensitivity is also enhanced although the configuration is easily obtained at low cost in the above-described manner.
  • a light component having the desired specific wavelength e.g. fluorescence
  • an unnecessary light component e.g. stray light of scattered light
  • the phosphor in the phosphor component is excited and the phosphor component emits fluorescence due to side scattered light of a precipitated substance of the pyrophosphoric acid generated in nucleic acid amplification and a metallic salt.
  • the fluorescence component can be measured without utilizing a fluorescent substance (fluorescent probe) of a nucleic acid amplification reaction solution.
  • the basis with an initial value 0% is easily set.
  • monitoring of the initial stage is easily performed for the user.
  • a filter e.g. fluorescent (wavelength selective transmissive) filter for noise removal
  • the phosphor component may be formed as plural layers in the sidewall part 21 (see e.g. phosphor components 231 and 232 in FIG. 4C ).
  • the plural phosphor layers By providing the plural phosphor layers, removing an unnecessary light component in advance is also permitted. Furthermore, the number of filters to remove noise in the device can be reduced. Therefore, the detection sensitivity is enhanced and size slimming of the device itself is also enabled. Furthermore, for example the following effect is achieved by forming plural different layers at certain intervals as shown in FIG. 4C . Specifically, after side scattered light from the reaction area is transmitted through the phosphor component 231 and becomes a fluorescence component, part of the fluorescence component is guided to the light detecting unit 5 by the reflective component. The remaining fluorescence component further passes through the phosphor component 232 and becomes a different fluorescence component. Thereafter, the fluorescence component is guided to the light detecting unit 5 by the reflective component.
  • each of different fluorescence components it is also possible to guide each of different fluorescence components to the light detecting unit 5 .
  • the number of fluorescent (wavelength selective transmissive) filters in the device can be reduced and therefore size reduction of the device itself is also permitted.
  • the material of the phosphor used for the phosphor component a publicly-known phosphor material may be used depending on the desired fluorescence component (about 300 to 750 nm).
  • the phosphor material either an organic phosphor or an inorganic phosphor may be employed.
  • an inorganic phosphor is preferable because the cost can be easily lowered and the desired wavelength selection can be easily achieved.
  • Various kinds of inorganic phosphors and organic phosphors will be exemplified below.
  • the phosphor material is not limited thereto.
  • the following materials can be used as the inorganic phosphor material. Any of them may be used solely or two or more kinds of them may be used in combination arbitrarily.
  • a phosphor composed of sialon (Si—Al—O—N) as the base particularly a fluorescent material that is composed mainly of ⁇ -sialon activated by Eu and is obtained by adding an element such as Ca, Y, or Mg to this ⁇ -sialon, is cited (refer to e.g. Japanese Patent Laid-open No. 2009-108223).
  • a fluorescent material composed of ⁇ -sialon having a different structure as the base, an inorganic compound having the same crystalline structure as that of the CaSiAlN 3 crystal, and a fluorescent material having the same crystalline structure as that of A 2 Si 5 N 8 are also cited.
  • These fluorescent materials have an advantage that white is easily obtained by emitting red and green with a blue LED (light-emitting diode) used as the light source.
  • Oxide phosphor materials composed of a garnet-based Y 3 Al 5 O 12 as the base are cited.
  • a fluorescent material represented by (Rel-rSmr) 3 (All-sGas) 5 O 12 : Ce (0 ⁇ r ⁇ 1, 0 ⁇ s ⁇ 1, Re is at least one kind of element selected from Y and Gd) as a general expression is cited (refer to e.g. Japanese Patent. Laid-open No. 2009-135545).
  • a Ca—Al—Si—O—N-based material doped with an Eu ion and oxynitride glass are cited (refer to e.g. Japanese Patent Laid-open No. 2008-227550).
  • an oxynitride-based fluorescent material a phosphor obtained by adding a group-V element to a phosphor based on the garnet structure, and red-added yellow phosphor and yellow-green phosphor obtained by adding Eu as an activator agent to an oxide of e.g. Ga, Al, or In and sulfurizing part of the oxide are cited.
  • Sialon phosphors for a white LED such as yellow “ ⁇ -sialon” and green “ ⁇ -sialon” are cited (refer to e.g. Japanese Patent Laid-open No. 2010-116564).
  • a characteristic of the ⁇ -sialon is that change in the luminance and the color with respect to temperature rise is smaller compared with a silicate-based green phosphor.
  • a light conversion material composed of a solidified body formed through continuous, three-dimensional intertwining of different metal oxides (e.g. Al 2 O 3 and Y 3 Al 5 O 12 ) with each other is cited (refer to e.g. Japanese Patent Laid-open No. 2006-173433).
  • a composite material obtained by precipitation of a YAG crystal in amorphous YAG is cited (refer to e.g. Japanese Patent Laid-open No. 2008-231218).
  • a semiconductor nanocrystal of CdS or the like and a composite body of a nanocrystal and a metal oxide are cited (refer to e.g. Japanese Patent Laid-open No. 2010-114079).
  • a material obtained by dispersing a semiconductor nanocrystal of ZnS or the like in a polymer matrix is cited (refer to e.g. JP-T-2010-528118).
  • a dielectric phosphor powder obtained by mixing a dielectric particle that does not absorb LED light of blue and so forth (particle with a wide band gap, AlN, air bubble, or the like) and a fluorescent (phosphorescent) material is cited (refer to e.g. Japanese Patent Laid-open No. 2002-261328).
  • organic phosphor material examples include the following molecular structure low-molecular series, metal complexes, polymer series, ⁇ -conjugated polymer materials, ⁇ -conjugated polymer materials, low-molecular-dye-containing polymer-based materials, and dopants. Any of them may be used solely or two or more kinds of them may be used in combination arbitrarily.
  • Examples of the molecular structure low-molecular series include distyrylbiphenyl-based blue luminescent material, dimesitylboryl-group-coupled amorphous luminescent material, stilbene-based conjugated dendrimer luminescent material, dipyridyl dicyanobenzene luminescent material, methyl-substituted benzoxazole-based fluorescence and phosphorescence emitting material, distyryl-based red luminescent material, heat-resistant carbazole-based green luminescent material, dibenzochrysene-based blue-green luminescent material, arylamine-based luminescent material, pyrene-substituted oligothiophene-based luminescent, material, divinylphenyl-coupled triphenylene-based luminescent material, perylene-based red luminescent material, PPV oligomer-based luminescent material, (carbazole-cyanoterephthalylidene)-based lumin
  • the metal complex examples include oxadiazole-beryllium blue luminescent complex, europium-based phosphorescence emitting complex, heat-resistant lithium-based blue luminescent complex, phosphorescence emitting phosphine-gold complex, terbium-based luminescent complex, thiophene-aluminum yellow luminescent complex, zinc-based yellow-green luminescent complex, amorphous aluminum-based green luminescent complex, boron-based luminescent complex, terbium-substituted europium-based luminescent complex, magnesium-based luminescent complex, phosphorescence emitting lanthanide-based near-infrared emitting complex, ruthenium-based luminescent complex, and copper-based phosphorescence emitting complex.
  • Examples of the polymer series include oligophenylenevinylene tetramer luminescent material.
  • ⁇ -conjugated polymer material examples include liquid-crystalline fluorene-based blue polarized light emitting polymer, binaphthalene-containing luminescent polymer, disilanyleneoligothienylene-based luminescent polymer, (fluorene-carbazole)-based blue luminescent copolymer, (dicyanophenylenevinylene-PPV)-based luminescent copolymer, silicon blue luminescent copolymer, conjugated chromophore group-containing luminescent polymer, oxadiazole-based luminescent polymer, PPV-based luminescent polymer, (thienylene-phenylene)-based luminescent copolymer, liquid-crystalline chiral-substituted fluorene-based blue luminescent polymer, spirofluorene-based blue luminescent polymer, thermally-stable diethylbenzene-based luminescent polymer, (binaphthyl-fluor
  • Examples of the ⁇ -conjugated polymer material include polysilane-based luminescent polymer.
  • Examples of the low-molecular dye-containing polymer-based material include carbazole side chain-coupled PMMA-based luminescent polymer and polysilane/dye-based luminescent composition.
  • Examples of the dopant include Eu complex-doped phosphorescence emitting material, triallylpyrazoline dopant compound, coronene-doped PVK luminescent material, thiophene-based compound-doped (PVK/PBD) luminescent material, Ir complex-doped PVK-based luminescent material, dipyrazole pyridine-based compound-doped luminescent material, pyran-based compound-doped Alq3 luminescent material, reduced porphyrin-doped Alq3 luminescent material, coumarin- or quinacridone-doped Alq-based luminescent material, ammonium salt-doped PVCz-based luminescent polymer, bithiophene-based compound-doped benzimidazole-based luminescent material, (butadiene-based compound: TPA) Co-doped PVK-based luminescent material, dye (TTP: DCM) Co-doped Alq3 luminescent material
  • the reaction area 2 be formed singularly or plurally in a reaction container (e.g. substrate 6 ) of a microchip for nucleic acid amplification reaction or the like for example.
  • the reaction container includes at least the reaction area 2 and the reflective component 20 (reflective surface 201 ), and it is preferable that the reaction container include the sidewall part 21 (sidewall 22 ) and the phosphor component 23 according to need.
  • the respective components be disposed around each reaction area 2 in the above-described order, i.e. in the order of the sidewall part 21 (sidewall 22 ), the phosphor component 23 , and the reflective component 20 (reflective surface 201 ), from the side of the reaction area 2 (see FIGS. 2A to 4C ).
  • the method for forming the nucleic acid amplification reaction microchip (substrate 6 ) including the reaction area 2 and the reflective component 20 is not particularly limited.
  • reaction area 2 it is preferable to form the reaction area 2 in the substrate e.g. by wet etching or dry etching of a glass substrate, layer or nanoimprint, injection forming, or cut processing of a plastic substrate layer.
  • the formed reaction area 2 may be filled with reagents for a nucleic, acid amplification reaction in advance.
  • the reflective component 20 in the substrate 6 e.g. by forming an inclined surface around the reaction area 2 and depositing a metal film on this surface by sputtering.
  • the material of the substrate 6 is not particularly limited and it is preferable to accordingly select the material in consideration of the detecting method, the processing easiness, the endurance, and so forth.
  • this material a material having optical transparency can be arbitrarily selected depending on the desired detecting method.
  • the material include glass and various kinds of plastic (polypropylene, polycarbonate, cycloolefin polymer, polydimethylsiloxane (PDMS), etc.).
  • the method for manufacturing the substrate 6 (micro flow path chip) of the embodiment of the present disclosure will be described in detail below based on the following procedures (A) to (G) (process flow). These procedures are one example of the method for fabricating the substrate 6 of the embodiment of the present disclosure and the manufacturing method is not limited thereto.
  • a transparent resin 30 e.g. SU8 photosensitive resin for forming the reflective component, serving as the mold of the micro flow path chip (substrate 6 ), is used (see FIG. 5A ).
  • a cylindrical structure to provide a well is fabricated into any shape by photolithography with use of the transparent resin 30 (see FIG. 5B ).
  • a transparent resin having an inclined surface is formed (see FIG. 5C ).
  • the transparent resin (resin mold 31 ) like that shown in FIG. 5C is used as the mold of the substrate 6 (reaction area 2 ).
  • a mixture solution of a transparent resin 62 (e.g. PDMS) is cast and cured on a substrate 61 (e.g. glass plate) based on the resin mold 31 , and the mold is released by separation (see FIG. 5D ).
  • a transparent resin 62 e.g. PDMS
  • a substrate 61 e.g. glass plate
  • a resist pattern having a predetermined circular shape is formed on the reflective component 20 by lithography and the reflective component 20 (metal film) is etched with use of this resist pattern as the mask (see FIG. 5E ). Thereby, the reflective component 20 (circular reflective film having an Ag/Au structure) is formed on the inclined surface over the substrate 61 (transparent resin).
  • the phosphor component 23 (sidewall) may be formed by applying a resin with which a phosphor material is mixed to the sidewall of the sidewall part 21 (not shown).
  • the whole of the sidewall part 21 may be formed as the phosphor component by mixing a phosphor material into the above-described mixture solution cast in the forming of the well shape.
  • a substrate 63 e.g. glass or plastic is so disposed that the space serving as the reaction area 2 is formed.
  • the micro flow path chip (substrate 6 ) having the reaction area 2 according to the embodiment of the present disclosure is obtained.
  • a substrate having a reflective component for reflection into the light incident surface direction can be Obtained by fabricating the substrate according to the above-described method and turning over the substrate after its completion for example.
  • Examples of the method for manufacturing the above-described resin mold 31 include, but not particularly limited to, the following methods (see FIGS. 6A to 6F ).
  • the inclined surface is automatically set to an angle of ⁇ 2 by applying a transparent resin over the whole surface by spin-coating.
  • the inclined surface is set to the angle of ⁇ 2 by applying a transparent resin by e.g. spin-coating and then curing and shrinking this transparent resin.
  • a transparent resin is formed by a photolithography technique. Specifically, the inclined surface is set to the angle of ⁇ 2 by using a resist (photosensitive resin) as the transparent resin and performing application, exposure, and development of this resist.
  • a resist photosensitive resin
  • the inclined surface is set to the angle of ⁇ 2 by press molding of a transparent resin with use of a predetermined mold.
  • the inclined surface is set to the angle of ⁇ 2 by thermal imprint of a transparent resin.
  • the inclined surface is set to the angle of ⁇ 2 by UV imprint molding of a transparent resin.
  • the inclined surface is set to the angle of ⁇ 2 by applying a transparent resin by e.g. spin-coating and then curing this transparent resin while this transparent resin is pressed against an elastically-deformable mold release layer.
  • nucleic acid amplification reaction includes existing polymerase chain reaction (PCR) in which a temperature cycle is implemented and various kinds of isothermal amplification methods involving no temperature cycle.
  • isothermal amplification method include loop-mediated isothermal amplification (LAMP) method, smart amplification process (SMAP) method, nucleic acid sequence-based amplification (NASBA) method, isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) method (registered trademark), transcription-reverse transcription concerted (TRC) method, strand displacement amplification (SDA) method, transcription-mediated amplification (TMA) method, and rolling circle amplification (RCA) method.
  • LAMP loop-mediated isothermal amplification
  • SMAP smart amplification process
  • NASBA nucleic acid sequence-based amplification
  • ICAN isothermal and chimeric primer-initiated amplification of nucleic acids
  • TRC transcription-reverse transcription concerted
  • SDA
  • nucleic acid amplification reaction widely encompasses nucleic acid amplification reactions based on a temperature-varying or isothermal process for the purpose of amplification of a nucleic acid. Furthermore, these nucleic acid amplification reactions encompass also reactions accompanied by quantification of the amplified nucleic acid strand, such as real-time PCR (RT-PCR) method and RT-LAMP method.
  • RT-PCR real-time PCR
  • Reagent includes reagents used to obtain an amplified nucleic acid strand in the above-described nucleic acid amplification reaction, specifically oligonucleotide primer with a base sequence that is complementary with the target nucleic acid strand, nucleic acid monomer (dNTP), enzyme, and reaction buffer solution (buffer) solute.
  • dNTP nucleic acid monomer
  • buffer buffer solution
  • the above-described LAMP method is a method in which dsDNA is obtained as the amplification product from DNA and RNA at a constant temperature by utilizing loop forming of the DNA.
  • the following components (i), (ii), and (iii) are added and the process proceeds through incubation at a temperature at which the inner primer can form base paring that is stable for the complementary sequence on the template nucleic acid and the strand-displacing polymerase can keep the enzyme activity. It is preferable that the incubation temperature be 50 to 70° C. and the time be about one minute to 10 hours.
  • component (i) two kinds of inner primers, or further two kinds of outer primers, or further two kinds of loop primers; component (ii) strand-displacing polymerase; component (iii) substrate nucleotide
  • Examples of the method for detecting the above-described nucleic acid amplification include a method of using a turbidity substance and a method of using a fluorescent substance or chemiluminescence substance.
  • Examples of the method of using a turbidity substance include a method of using a precipitated substance generated due to the pyrophosphoric acid resulting from the nucleic acid amplification reaction and a metal ion that can be coupled to it.
  • This metal ion is a monovalent or divalent metal ion.
  • When being coupled to the pyrophosphoric acid it forms a salt that is insoluble or poorly-soluble in water and becomes the turbidity substance.
  • the metal ion include alkali metal ion, alkaline earth metal ion, and divalent transition metal ion.
  • one or more kinds of metal ions selected from alkaline earth metal ions such as magnesium (II), calcium (II), and barium (II); and divalent transition metal ions such as zinc (II), lead (II), manganese (II), nickel (II), and iron (II) are preferable.
  • alkaline earth metal ions such as magnesium (II), calcium (II), and barium (II); and divalent transition metal ions such as zinc (II), lead (II), manganese (II), nickel (II), and iron (II) are preferable.
  • Magnesium (II), manganese (II), nickel (II), and iron (II) are particularly preferable.
  • the concentration of the added metal ion be in the range of 0.01 to 100 mM. It is preferable to set the detection wavelength to 300 to 800 nm.
  • Examples of the method of using a fluorescent substance or a chemiluminescence substance include an intercalate method of using a fluorescent dye (derivative) that is specifically intercalated into a double-stranded nucleic acid and emits fluorescence, and a labeled probe method of using a probe obtained by coupling a fluorescent dye to oligonucleotide that is specific to the nucleic acid sequence to be amplified.
  • Examples of the labeled probe method include hybridization (Hyb) probe method and hydrolysis (TaqMan) probe method.
  • the Hyb probe method is a method of using two kinds of probes, i.e. a probe labeled with a donor dye that is so designed that two kinds of probes get close to each other in advance, and a probe labeled with an acceptor dye.
  • a probe labeled with a donor dye that is so designed that two kinds of probes get close to each other in advance
  • an acceptor dye When these two kinds of probes are hybridized with the target nucleic acid, the acceptor dye excited by the donor dye emits fluorescence.
  • the TaqMan probe method is a method of using a probe that is so labeled that a reporter dye and a quencher dye get close to each other. This probe is hydrolyzed in nucleic acid extension. At this time, the quencher dye and the reporter dye get separated, and fluorescence is emitted in response to excitation of the reporter dye.
  • Examples of the fluorescent dye (derivative) used in the method of using a fluorescent substance include SYBR (registered trademark) Green I, SYBR (registered trademark) Green II, SYBR (registered trademark) Gold, YO (Oxazole Yellow), TO (Thiazole Orange), PG (Pico (registered trademark) Green), and ethidium bromide.
  • Examples of the organic compound used in the method of using a chemiluminescence substance include luminol, lophine, lucigenin, and oxalate.
  • the irradiating unit 3 may be any unit as long as it includes a light source 3 a and has such a configuration that light L 1 emitted from the light source is irradiated to the reaction area 2 .
  • the light source 3 a supported by a support body 3 b may be disposed above and/or below the reaction area 2 (see FIG. 1 ).
  • an optical guide component to guide the light L 1 emitted from the light source 3 a to the reaction area 2 may be disposed (not shown).
  • the irradiating unit 3 include the optical guide component.
  • a light incident end part is made in the optical guide component and light emitted from one or plural light sources 3 a is incident on the light incident end part.
  • Components e.g. prism, reflective plate, and concave and convex part
  • for guiding the incident light L to the respective reaction areas are provided inside the optical guide component.
  • the optical guide component By disposing the optical guide component, the number of light sources can be reduced and uniform light can be irradiated to one or plural reaction areas 2 on the substrate 6 . Furthermore, the detection sensitivity and the detection accuracy in turbidity detection are also favorable. In addition, due to the reduction in the number of light sources, size reduction of the whole device, particularly thickness reduction, is also permitted and power consumption reduction is also enabled.
  • the light source 3 a is not particularly limited, a light source that emits the desired light allowing favorable detection of the target nucleic acid amplification product is preferable as the light source 3 a .
  • the light source 3 a include laser light source, white or single-color light emitting diode (LED), mercury lamp, and tungsten lamp.
  • the LED is preferable because it allows power consumption reduction and cost reduction.
  • the LED is advantageous because it also enables achievement of the desired light component if various kinds of filters are used.
  • the laser light source is not particularly limited by the kind of laser light.
  • a light source that emits e.g. argon (Ar) ion laser, helium-neon (He—Ne) laser, dye laser, or krypton (Kr) laser is enough as the laser light source.
  • Ar argon
  • He—Ne helium-neon
  • dye laser dye laser
  • Kr krypton
  • one kind of laser light source may be used or two or more kinds of laser light sources may be used in combination freely.
  • the temperature control unit 4 is to heat the reaction area 2 .
  • Examples of the temperature control unit 4 include, but not particularly limited to, heater of a Peltier element or the like and ITO heater having optical transparency.
  • Examples of the shape of the temperature control unit 4 include thin film shape and flat plate shape.
  • the temperature control unit 4 be disposed at such a position that heat is easily transferred to the reaction area 2 .
  • the temperature control unit 4 be disposed close to the reaction area 2 .
  • it may be disposed at any of positions such as positions above, below, and beside the reaction area 2 and a position at the outer circumference of the reaction area 2 .
  • the temperature control unit 4 have a thin film or flat plate shape and be disposed above and/or below the reaction area 2 .
  • the temperature control unit 4 may be disposed as a substrate support mounting.
  • a hole may be made in the temperature control unit 4 so that light may pass through it. This eliminates the need to increase the distance from the heat source and thus facilitates temperature control inside the reaction area 2 . Therefore, the detection sensitivity and the detection accuracy are enhanced.
  • the light detecting unit 5 may be any unit as long as it is such a mechanism as to be capable of detecting the amount of light of light beams L 3 and L 4 (L 5 ) obtained by reflecting the side light from the reaction area 2 by the reflective component 20 .
  • the light detecting unit 5 is provided with at least an optical detector 5 a and this optical detector 5 a is accordingly supported by a support body 5 b . It is enough that each optical detector 5 a is so disposed as to correspond to guided light and the optical detectors 5 a are disposed one-dimensionally, two-dimensionally, or three-dimensionally for example.
  • optical detector 5 a examples include, but not limited to, area imaging elements such as photodiode (PD) array, CCD (Charge Coupled Device) image sensor, and CMOS (Complementary Metal Oxide Semiconductor) image sensor, small optical sensor, line sensor scan, and photomultiplier tube (PMT). Any of them may be combined arbitrarily.
  • area imaging elements such as photodiode (PD) array, CCD (Charge Coupled Device) image sensor, and CMOS (Complementary Metal Oxide Semiconductor) image sensor, small optical sensor, line sensor scan, and photomultiplier tube (PMT). Any of them may be combined arbitrarily.
  • PD photodiode
  • CCD Charge Coupled Device
  • CMOS Complementary Metal Oxide Semiconductor
  • An excitation filter and a fluorescent filter may be disposed in the nucleic acid amplification reaction device 1 of the embodiment of the present disclosure arbitrarily.
  • the excitation filter a light component having the desired specific wavelength can be obtained depending on the method for detecting a nucleic acid amplification reaction and an unnecessary light component can be removed.
  • the fluorescent filter light is turned to the light component (scattered light, transmitted light, and fluorescence) necessary for detection. This enhances the detection sensitivity and the detection accuracy.
  • nucleic acid amplification reaction method of detecting the amount of scattered light (the amount of fluorescence) due to a turbidity substance formed from the pyrophosphoric acid and a metal salt will be made below about a nucleic acid amplification reaction method of detecting the amount of scattered light (the amount of fluorescence) due to a turbidity substance formed from the pyrophosphoric acid and a metal salt.
  • ⁇ 1aA step> The light L 1 is emitted from the light source 3 a and becomes light L 2 (excited light) due to the excitation filter 8 .
  • This light L 2 is irradiated to the reaction area 2 serving as the reaction filed of an amplification reaction of a nucleic acid.
  • ⁇ 1aB step> At this time, a substance precipitated in the nucleic acid amplification reaction (turbidity substance) is generated and thereby the degree of light scattering increases.
  • the light L 2 is irradiated to the precipitated substance, generated in association with the progression of the nucleic acid amplification reaction in the reaction area 2 .
  • the scattered light L 3 and the side scattered light L 4 are generated from the precipitated substance in the reaction area 2 .
  • ⁇ 1aC step> The side scattered light L 4 is reflected by the reflective component 20 (reflective surface 201 ) disposed on the lateral side of the reaction area 2 and output in the light output surface direction.
  • the amount of light is detected by the light detecting unit 5 (optical detector 5 a ). That is, the amount of scattered light due to the precipitated substance generated in association with the progression of the amplification reaction is detected.
  • ⁇ 1bA step> This step is the same as the above-described ⁇ 1aA step>.
  • ⁇ 1bB step> This step is the same as the above-described ⁇ 1aB step>.
  • ⁇ 1bC step> The side scattered light L 4 from the reaction area 2 is transmitted through the phosphor component 23 (sidewall including a phosphor component, phosphor component layer, or the like) to thereby become fluorescence (light L 5 ).
  • This light L 5 is reflected by the reflective component 20 (reflective surface 201 ) disposed on the lateral side of the reaction area 2 and output in the light output surface direction.
  • the output light L 5 is detected by the light detecting unit 5 (optical detector 5 a ) as the amount of light. That is, the precipitated substance generated in association with the progression of the amplification reaction is detected based on the amount of fluorescence.
  • the scattered light L 3 is the same as that in the above-described case (1-a).
  • nucleic acid amplification reaction method of detecting a fluorescent substance generated in a nucleic acid amplification reaction With reference to FIG. 1 and FIG. 2A , a description will be made below about a nucleic acid amplification reaction method of detecting a fluorescent substance generated in a nucleic acid amplification reaction.
  • ⁇ 2aA step> This step is the same as the above-described ⁇ 1aA step>.
  • ⁇ 2aB step> The light L 2 is irradiated to a fluorescent substance generated in association with the progression of a nucleic acid amplification reaction in the reaction area 2 . At this time, the amount of fluorescence increases due to the generation of the fluorescent substance in the nucleic acid amplification reaction. Accordingly, the forward fluorescence L 3 and the side fluorescence L 4 are generated from the fluorescent substance in the reaction area 2 .
  • ⁇ 2aC step> The light L 4 is reflected by the reflective component 20 (reflective surface 201 ) disposed on the lateral side of the reaction area 2 and output in the light output surface direction.
  • the output light L 4 is detected by the light detecting unit 5 (optical detector 5 a ) as the amount of light. That is, the amount of fluorescence due to the fluorescent substance generated in association with the progression of the amplification reaction is detected.
  • ⁇ 2bA step> This step is the same as the above-described ⁇ 1aA step>.
  • ⁇ 2bB step> The light L 4 from the reaction area 2 is transmitted through the phosphor component 23 (sidewall including a phosphor component, phosphor component layer, or the like) to thereby become a fluorescence component having a specific wavelength (light L 5 ).
  • This light L 5 is reflected by the reflective component 20 (reflective surface 201 ) disposed on the lateral side of the reaction area 2 and output in the light output surface direction.
  • the amount of output light is detected by the light detecting unit 5 (optical detector 5 a ). That is, the fluorescent substance generated in association with the progression of the amplification reaction is detected based on the amount of fluorescence of the light component having the specific wavelength.
  • the fluorescence L 3 is the same as that in the above-described case (2-a).
  • FIG. 7 is a schematic conceptual diagram schematically showing a nucleic acid amplification reaction device 1 according to a second embodiment of the present disclosure. Description of the same configuration as that in the first embodiment is omitted.
  • the nucleic acid amplification reaction device 1 includes at least the detachable substrate 6 having the reaction area 2 and the reflective component 20 , the irradiating unit 3 , and the light detecting unit 5 , and may include the temperature control unit 4 arbitrarily.
  • the light detecting unit 5 is disposed between the irradiating unit 3 and the reaction area 2 (substrate 6 ).
  • the excitation filter 8 and the collecting lens 9 may be disposed between the light detecting unit 5 and the irradiating unit 3 arbitrarily. Furthermore, the collecting lens 11 and the fluorescent filter 10 may be disposed between the light detecting unit 5 and the reaction area 2 arbitrarily.
  • a light detecting unit 51 may be disposed on the light output surface side of the reaction area 2 and a fluorescent filter (not shown) may be provided between the reaction area 2 and the light detecting unit 51 . This allows detection for initialization of the initial value of irradiated light and enhances the detection sensitivity, particularly the detection sensitivity from the timing of the reaction start.
  • a substrate support mounting (temperature control unit 4 ) may be disposed on the light incident surface side of the reaction area 2 arbitrarily.
  • the substrate 6 shown in FIG. 2B is preferable as the substrate 6 (micro flow path chip) mounted in the above-described nucleic acid amplification reaction device 1 (second embodiment).
  • the side light from the reaction area 2 is reflected by the reflective component 20 (reflective surface 201 ) to thereby be output in the light incident surface direction by return.
  • the light L 1 from the irradiating unit 3 is transmitted through the excitation filter 8 and becomes the light L 2 .
  • This light L 2 is transmitted through the support body 5 b supporting, the light detecting unit 5 and further transmitted through the temperature control unit 4 (substrate support mounting) to be irradiated to the reaction area 2 .
  • the light L 2 is irradiated to the nucleic acid amplification product in the reaction area 2 and the light L 4 generated toward the lateral side is reflected by the reflective component 20 (reflective surface 201 ) into the light incident surface direction.
  • This reflected light L 4 passes through the temperature control unit 4 and goes through the collecting lens 11 and then the fluorescent filter 10 , so that the light component is detected by the light detecting unit 5 .
  • the reaction area 2 after the reaction end can be set on e.g. the temperature control unit 4 and used also as a nucleic acid amplification detecting device.
  • the substrate 6 (micro flow path chip) of the embodiment of the present disclosure in a LAMP device and a PCR device and quantify a nucleic acid with use, of a fluorescent substance or a turbidity substance in the reaction area as an index.
  • the operation of these devices when a turbidity substance is used as an index will be described below.
  • a temperature control step (step S 11 ), the temperature is so set that a constant temperature (60 to 65° C.) is kept in the reaction area 2 , and thereby a nucleic acid in each reaction area 2 is amplified.
  • a constant temperature 60 to 65° C.
  • thermal denaturation from a single-stranded nucleic acid to a double-stranded nucleic acid is unnecessary and primer annealing and nucleic acid extension are repeatedly performed under this isothermal condition.
  • the pyrophosphoric acid is generated and a metal ion is coupled to this pyrophosphoric acid, so that an insoluble or poorly-soluble salt is formed and this salt acts as a turbidity substance (measurement wavelength 300 to 800 nm).
  • the incident light (light L) is irradiated to this turbidity substance to thereby become scattered light (light L 1 , L 2 ).
  • the amount of scattered light is measured by the light detecting unit 5 in real time to be quantified. Quantification from the amount of transmitted light is also possible. If the phosphor component 23 is present in the substrate, quantification from the amount of fluorescence is possible.
  • the substrate includes the phosphor component 23 , light can be turned to a specific fluorescence component and quantification from the amount of fluorescence of this specific fluorescence component is permitted.
  • step Sp 1 thermal denaturation
  • step Sp 2 primary annealing
  • step Sp 3 DNA extension
  • step Sp 1 the temperature is so controlled by the temperature control unit that 95° C. is kept in the reaction area 2 and a double-stranded DNA is turned to a single-stranded DNA through denaturation.
  • step Sp 2 the temperature is so set that 55° C. is kept in the reaction area 2 .
  • the primer is coupled to the base sequence that is complementary with this single-stranded DNA.
  • step Sp 3 the temperature is so controlled that 72° C. is kept in the reaction area 2 . Thereby, by use of the primer as the start point of DNA synthesis, the polymerase reaction is progressed to extend the cDNA.
  • the DNA in each reaction area 2 is amplified.
  • the pyrophosphoric acid is generated and a turbidity substance is detected in the above-described manner, so that the amount of nucleic acid is quantified as described above. If a phosphor component is present in the substrate, quantification from the amount of fluorescence is possible.
  • the substrate includes the phosphor component 23 , light can be turned to a specific fluorescence component and quantification from the amount of fluorescence of this specific fluorescence component is permitted.
  • the side light that is generated due to light irradiation and is from the reaction area serving as the reaction field of a nucleic acid amplification reaction be guided into the light output surface direction and/or the light incident surface direction by the reflective component disposed around this reaction area and the amount of guided light be detected by the light detector. Furthermore, it is preferable that the side light be side scattered light and the amount of fluorescence arising from transmission of this side scattered light through a phosphor component be detected. This makes it possible to easily extract scattered light and fluorescence accordingly. Thus, nucleic acid amplification can be measured easily with higher sensitivity. In addition, the need to use an expensive organic fluorescent probe is eliminated. Therefore, measurement at low cost is permitted and quality retention of reagents is enhanced. Moreover, there is an advantage that using the method causes a trouble neither in reaction detection by the related-art turbidity detection nor in optical detection by an organic fluorescent probe.
  • a cylindrical structure to provide a well was fabricated into any shape by photolithography with use of an SU8 photosensitive resin serving as the mold of a micro flow path chip.
  • the inclined surface was automatically set to an angle of ⁇ 2 by applying a transparent resin over the whole surface by spin-coating.
  • a mixture solution of PDMS was cast and cured based on the above-described fabricated mold, and the mold was released by separation.
  • a via serving as the well and an inclined surface on the circumference of the via were formed on the PDMS resin from which the mold was released.
  • Ag film and Au film were sequentially formed over the whole surface of the PDMS substrate by e.g. sputtering.
  • a resist pattern having a predetermined circular shape was formed thereon by lithography and the Ag film and Au film were etched with use of this resist pattern as the mask.
  • a circular reflective film having an Ag/Au structure was formed on the transparent resin.
  • the material of the reflective film a substance having extremely high reflectance to light of the emission wavelength, e.g. Ag or a metal composed mainly of Ag, was used. This is because this allows end surface emitted light and circulated light returned through reflection by the glass/PDMS to be efficiently reflected by this reflective film and the light is easily extracted to the outside finally.
  • a primer solution for LAMP was mixed.
  • the design of the LAMP method primer was carried out by utilizing six domains, i.e. F3 domain, domain, F1 domain, B1 domain, B2 domain, and B3 domain, from the 5′ side of the target sequence.
  • six domains i.e. F3 domain, domain, F1 domain, B1 domain, B2 domain, and B3 domain, from the 5′ side of the target sequence.
  • four kinds of primers are used.
  • the inner primers couple F1c to F2 and couple B1c to B2.
  • the forward loop primer was set for the complementary strand for the domain between F1 domain and F2 domain
  • the backward loop primer was set for the complementary strand for the domain between B1 domain and B2 domain.
  • the free energy of the 3′ end of F2/B2, F3/B3, and LF/LB and the 5′ end of F1c/B1c was set equal to or lower than ⁇ 4 kcal/mol.
  • FIP-BIP and F3 and B3 domains were designed from the whole target domain and subsequently a primer set obtained by selecting and combining one pair of F3 and B3 domains was designed for each FIP-BIP domain.
  • the combination of FIP-BIP and F3 and B3 domains starts from the 5′ end and continues to the 3′ end. Subsequently, the combination starts from the 5′ end again and the design is progressed toward the 3′ end, and at most three kinds of F3-B3 are combined for one FIP-BIP.
  • the codes of the primer designed by Primer Explorer are shown in the following Table 1.
  • the PDMS substrate in which enzyme and primer were immobilized to all wells was subjected to DP ashing under a condition of O 2 : 10 cc, 100 W, and 30 seconds to turn the surface to a hydrophilic surface. Then the PDMS substrate was bonded to a cover glass in vacuum.
  • the PDMS was penetrated by a painless needle and an extraction mixture solution for reaction with a quantified copy number was introduced into the flow path in the chip.
  • the chip was set in a fluorescence detecting device including a fluorescence detector and a heater over a measurement substrate for each reaction area (well). In this device, excitation light from an LED was irradiated from above each well in the microchip substrate simultaneously with the reaction and light scattered by a reaction by-product in the reaction area was detected.
  • the excitation light scattered in the well was irradiated to an inorganic phosphor of the well sidewall and fluorescence was emitted.
  • This fluorescence was detected and measured by a fluorescence detecting photodetector provided below the microchip substrate reaction area disposed on the optical axis of the excitation light source.
  • the S/N ratio is low and the determination is difficult until the particular size of the magnesium pyrophosphate colloid becomes sufficiently large and white turbidity is caused.
  • the projected surface area was increased by extracting laterally scattered light.
  • a sufficiently high S/N ratio could be ensured because almost no side scattering is present in a transmissive optical system including no scattering object.
  • the S/N ratio with respect to incident light could be enhanced because a light receiver could be disposed on the incident light side and a filter could be applied to the light receiver if fluorescence was obtained by side scattered light.
  • the nucleic acid amplification reaction device can ensure a sufficiently-high. S/N ratio by reflecting the side light by the reflective component, and allows measurement with high detection sensitivity although it is easy to use. In addition, by using the phosphor component, removal of an unnecessary light component and achievement of a specific light component are also permitted. Thus, despite low cost, easy measurement with high detection sensitivity is possible.

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